Debaryomyces hansenii, Stenotrophomonas rhizophila, and Ulvan as Biocontrol Agents of Fruit Rot Disease in Muskmelon (Cucumis melo L.)

The indiscriminate use of synthetic fungicides has led to negative impact to human health and to the environment. Thus, we investigated the effects of postharvest biocontrol treatment with Debaryomyces hansenii, Stenotrophomonas rhizophila, and a polysaccharide ulvan on fruit rot disease, storability, and antioxidant enzyme activity in muskmelon (Cucumis melo L. var. reticulatus). Each fruit was treated with (1) 1 × 106 cells mL−1 of D. hansenii, (2) 1 × 108 CFU mL−1 of S. rhizophila, (3) 5 g L−1 of ulvan, (4) 1 × 106 cells mL−1 of D. hansenii + 1 × 108 CFU mL−1 of S. rhizophila, (5) 1 × 108 CFU mL−1 of S. rhizophila + 5 g L−1 of ulvan, (6) 1 × 106 cells mL−1 of D. hansenii + 1 × 108 CFU mL−1 of S. rhizophila + 5 g L−1 of ulvan, (7) 1000 ppm of benomyl or sterile water (control). The fruits were air-dried for 2 h, and stored at 27 °C ± 1 °C and 85–90% relative humidity. The fruit rot disease was determined by estimating the disease incidence (%) and lesion diameter (mm), and the adhesion capacity of the biocontrol agents was observed via electron microscopy. Phytopathogen inoculation time before and after adding biocontrol agents were also recorded. Furthermore, the storability quality, weight loss (%), firmness (N), total soluble solids (%), and pH were quantified. The antioxidant enzymes including catalase, peroxidase, superoxide dismutase, and phenylalanine ammonium lyase were determined. In conclusion, the mixed treatment containing D. hansenii, S. rhizophila, and ulvan delayed fruit rot disease, preserved fruit quality, and increased antioxidant activity. The combined treatment is a promising and effective biological control method to promote the shelf life of harvested muskmelon.

Exploitation of Yeasts with Probiotic Traits for Kefir Production: Effectiveness of the Microbial Consortium

Kefir is a fermented milk made by beneficial lactic acid bacteria and yeasts inoculated as grains or free cultures. In this work, five yeast strains with probiotic aptitudes belonging to Candida zeylanoides, Yarrowia lipolytica, Kluyveromyces lactis, and Debaryomyces hansenii species were assessed in a defined consortium, in co-culture with a commercial strain of Lactobacillus casei, in order to evaluate the yeasts’ fermentation performance during kefir production, using different milks. The concentration of each yeast was modulated to obtain a stable consortium that was not negatively affected by the bacteria. Furthermore, all yeasts remained viable for five weeks at 4 °C, reaching about 8.00 Log CFU in 150 mL of kefir, a volume corresponding to a pot of a commercial product. The yeasts consortium showed a suitable fermentation performance in all milks, conferring peculiar and distinctive analytical and aromatic properties to the kefirs, confirmed by a pleasant taste. Overall, the panel test revealed that the cow’s and sheep’s kefir were more appreciated than the others; this evaluation was supported by a distinctive fermentation by-products’ content that positively influences the final aroma, conferring to the kefir exalted taste and complexity. These results allow us to propose the yeasts consortium as a versatile and promising multistarter candidate able to affect industrial kefir with both recognizable organoleptic properties and probiotic aptitudes.

Several Yeast Species Induce Iron Deficiency Responses in Cucumber Plants (Cucumis sativus L.)

Iron (Fe) deficiency is a first-order agronomic problem that causes a significant decrease in crop yield and quality. Paradoxically, Fe is very abundant in most soils, mainly in its oxidized form, but is poorly soluble and with low availability for plants. In order to alleviate this situation, plants develop different morphological and physiological Fe-deficiency responses, mainly in their roots, to facilitate Fe mobilization and acquisition. Even so, Fe fertilizers, mainly Fe chelates, are widely used in modern agriculture, causing environmental problems and increasing the costs of production, due to the high prices of these products. One of the most sustainable and promising alternatives to the use of agrochemicals is the better management of the rhizosphere and the beneficial microbial communities presented there. The main objective of this research has been to evaluate the ability of several yeast species, such as Debaryomyces hansenii, Saccharomyces cerevisiae and Hansenula polymorpha, to induce Fe-deficiency responses in cucumber plants. To date, there are no studies on the roles played by yeasts on the Fe nutrition of plants. Experiments were carried out with cucumber plants grown in a hydroponic growth system. The effects of the three yeast species on some of the most important Fe-deficiency responses developed by dicot (Strategy I) plants, such as enhanced ferric reductase activity and Fe2+ transport, acidification of the rhizosphere, and proliferation of subapical root hairs, were evaluated. The results obtained show the inductive character of the three yeast species, mainly of Debaryomyces hansenii and Hansenula polymorpha, on the Fe-deficiency responses evaluated in this study. This opens a promising line of study on the use of these microorganisms as Fe biofertilizers in a more sustainable and environmentally friendly agriculture.

Bioprocesses with Reduced Ecological Footprint by Marine Debaryomyces hansenii Strain for Potential Applications in Circular Economy

The possibility to perform bioprocesses with reduced ecological footprint to produce natural compounds and catalyzers of industrial interest is pushing the research for salt tolerant microorganisms able to grow on seawater-based media and able to use a wide range of nutrients coming from waste. In this study we focused our attention on a Debaryomyces hansenii marine strain (Mo40). We optimized cultivation in a bioreactor at low pH on seawater-based media containing a mixture of sugars (glucose and xylose) and urea. Under these conditions the strain exhibited high growth rate and biomass yield. In addition, we characterized potential applications of this yeast biomass in food/feed industry. We show that Mo40 can produce a biomass containing 45% proteins and 20% lipids. This strain is also able to degrade phytic acid by a cell-bound phytase activity. These features represent an appealing starting point for obtaining D. hansenii biomass in a cheap and environmentally friendly way, and for potential use as an additive or to replace unsustainable ingredients in the feed or food industries, as this species is included in the QPS EFSA list (Quality Presumption as Safe—European Food Safety Authority).

Physicochemical Parameters Limiting Growth of Debaryomyces hansenii in Solutions of Hygroscopic Compounds and Their Effects on the Habitability of Martian Brines

The availability of liquid water is a prerequisite for all lifeforms on Earth. In hyperarid subzero environments like the Dry Valleys in Antarctica or the near-subsurface of Mars liquid water might be provided temporarily by hygroscopic substances that absorb water from the atmosphere and lower the freezing point of water. To evaluate the potential of hygroscopic compounds to serve as a habitat, it is necessary to explore the microbial tolerances towards these substances and their life-limiting properties. Here we present a study investigating the tolerances of the halotolerant yeast Debaryomyces hansenii to various solutes. Growth experiments were conducted via counting colony forming units (CFUs) after inoculation of a liquid growth medium containing a specific solute concentration. The lowest water activities (aw) enabling growth were determined to be ~0.83 in glycerol and fructose-rich media. For all other solutes the growth-enabling aw was higher, due to additional stress factors such as chaotropicity and ionic strength. Additionally, we found that the solute tolerances of D. hansenii correlate with both the eutectic freezing point depressions and the deliquescence relative humidities of the respective solutes. Our findings strongly impact our understanding of the habitability of solute-rich low aw environments on Earth and beyond.

A CRISPR/Cas9 Method Facilitates Efficient Oligo-Mediated Gene Editing in Debaryomyces Hansenii

The halophilic and osmotolerant yeast Debaryomyces hansenii has a high potential for cell factory applications due to its resistance to harsh environmental factors and compatibility with a wide substrate range. However, currently available genetic techniques does not allow the full potential of D. hansenii as a cell factory to be harnessed. Moreover, most of the currently available tools rely on the use of auxotrophic markers that are not suitable in wild-type prototrophic strains. In addition, the preferred non-homologous end-joining (NHEJ) DNA damage repair mechanism pose further challenges when precise gene targeting is required. In this study, we present a novel plasmid based CRISPRCUG/Cas9 method for easy and efficient gene editing of the prototrophic strains of D. hansenii. Our toolset design is based on a dominant marker and facilitates quick assembly of the vectors expressing Cas9 and single or multiple sgRNAs that provides possibility for multiplex gene engineering even in prototrophic strains. Moreover, we have constructed an NHEJ deficient D. hansenii that enable our CRISPRCUG/Cas9 tools to support highly efficient introduction of point mutations and single/double gene deletions. Importantly, we also demonstrate that 90-nt single stranded DNA oligonucleotides are sufficient to direct repair of DNA breaks induced by sgRNA-Cas9 resulting in precise edits reaching 100% efficiencies. In conclusion, tools developed in this study will greatly advance basic and applied research in D. hansenii. In addition, we envision that our tools can be rapidly adapted for gene editing of other non-conventional yeast species including the ones belonging to the CUG clade.